Biology subjects

Nakamura, M.

Publications and source records attributed to Nakamura, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Identification of nuclear pore proteins at plasmodesmata

Plasmodesmata (PD) mediate intercellular exchange of small molecules, RNAs and proteins between plant cells with an apparent exclusion limit for passive non-specific transport, and transport of specific cargo mediated by mediators. PD and nuclear pore complexes (NPC) are nanometer sized micropores with strikingly similar properties. Cargo translocation through NPC is mediated by phase separating FG-nucleoporins (FG-NUP). Here, bioinformatics, proteomics and fluorescence imaging identified FG-NUPs at PD. Transient expression of GFP fusions at low and intermediate expression levels supported dual localization of 12 NUPs to NPC and PD. Structured illumination microscopy detected the transmembrane anchor NUP CPR5 close to orifices of PD. cpr5 mutants showed reduced intercellular short-root (SHR) transport. However, transport defects cannot be excluded due to indirect effects in the mutants. Identification of FG-NUPs at PD is consistent with the recruitment of NUPs to form a PD pore gating complex consistent with phase separation domains as diffusion barriers at PD. Further analyses will be required to determine whether NUPs are bona fide PD components, or accumulate at PD in certain conditions, or may serve intermediate NPC storage.

cell biology↗

bioRxiv2024

Bone mechano-response is driven by locomotion transitions during vertebrate evolution

The skeleton supports the muscles in keeping the body upright against gravity while enduring thousands of daily loads. In this study, we investigated non-collagenous bone matrix proteins using osteoblast cell cultures and phylogenetic analyses to identify the molecular mechanisms involved in mechanical loading. The results indicate that the bone mechano-response is an evolutionary-driven process and that several non-collagenous proteins may significantly regulate the bones response to mechanical stress. According to our results, two significant evolutionary transitions in vertebrate locomotion shaped the roles of non-collagenous proteins in humans: the water-to-land transition, which increased mechanical stress on the limbs, and the evolution to bipedalism in humans, which altered the distribution of stress on the lower and upper limbs. Fetuin A, positively selected in both evolutionary transitions, showed the most significant expression change during mechanical stimulation.

evolutionary biology↗

bioRxiv2024

Histone H2B isoform H2bc27 is expressed in the developing brain of mouse embryos

Histones bind directly to DNA and play a role in regulating gene expression mediated by chromatin structure. DNA sequences of these histone genes are quite similar, which has hindered individual analyses. The exact function of the 13 different isoforms of histone H2B remains unclear. In this report, a comprehensive gene expression analysis of the H2B isoforms, focusing on tissue specificity, was conducted. We generated mice lacking the H2bc27 gene, which exhibited brain-specific expression of E14.5, and proceeded to characterize the brain tissue. The phenotype of H2bc27 knockout brains was similar to that of wild-type brains, yet transcriptome analysis indicated that H2bc27 is associated with regulating the expression of several functional genes involved in mouse brain development. The methods used in this study may serve to facilitate comprehensive H2B isoform analysis.

developmental biology↗

bioRxiv2024

Computational Mechanisms of Neuroimaging Biomarkers Uncovered by Multicenter Resting-State fMRI Connectivity Variation Profile

Resting-state functional connectivity (rsFC) is increasingly used to develop biomarkers for psychiatric disorders. Despite progress, development of the reliable and practical FC biomarker remains an unmet goal, particularly one that is clinically predictive at the individual level with generalizability, robustness, and accuracy. In this study, we propose a new approach to profile each connectivity from diverse perspective, encompassing not only disorder-related differences but also disorder-unrelated variations attributed to individual difference, within-subject across-runs, imaging protocol, and scanner factors. By leveraging over 1500 runs of 10-minute resting-state data from 84 traveling-subjects across 29 sites and 900 participants of the case-control study with three psychiatric disorders, the disorder-related and disorder-unrelated FC variations were estimated for each individual FC. Using the FC profile information, we evaluated the effects of the disorder-related and disorder-unrelated variations on the output of the multi-connectivity biomarker trained with ensemble sparse classifiers and generalizable to the multicenter data. Our analysis revealed hierarchical variations in individual functional connectivity, ranging from within-subject across-run variations, individual differences, disease effects, inter-scanner discrepancies, and protocol differences, which were drastically inverted by the sparse machine-learning algorithm. We found this inversion mainly attributed to suppression of both individual difference and within-subject across-runs variations relative to the disorder-related difference by weighted-averaging of the selected FCs and ensemble computing. This comprehensive approach will provide an analytical tool to delineate future directions for developing reliable individual-level biomarkers.

neuroscience↗

bioRxiv2024

Identification of Large Japanese field mouse Apodemus speciosus food plant resources in an industrial green space using DNA metabarcoding

DNA metabarcoding was employed to identify the food plant resources of the Large Japanese field mouse Apodemus speciosus, inhabiting an artificial green space on reclaimed land on the Chita Peninsula in Aichi Prefecture, Central Japan, from 2012 to 2014. DNA metabarcoding was performed using high-throughput sequencing of partial rbcL sequences extracted from feces samples collected in the study area. The obtained sequences, which were analyzed using a constructed local database, revealed that a total of 72 plant taxa were utilized as food plant resources by A. speciosus. Of these plant taxa, 43 could be assigned to species (59.7%), 16 to genus (22.2%), and 13 to family (18.1%). Of the 72 plant taxa identified in this study, the dominant families throughout all collection periods were Lauraceae (81.0% of 100 fecal samples), followed by Fagaceae (70.0%), Rosaceae (68.0%), and Oleaceae (48.0%). Fifty of the 72 plant taxa identified as food plant resources were woody plants. An analysis employing rarefaction techniques for each season in the study site indicated comprehensive coverage of the food plant resources, ranging from 86.4% in winter to 93.6% in spring. Further, 96.5% of the food plant taxa were found throughout the study period. The findings showed that DNA metabarcoding using a local database constructed from the National Center for Biotechnology Information (NCBI) database and field surveys was effective for identifying the dominant food plants in the diet of A. speciosus. The results of this study provided basic information that can be applied to formulation and implementation of management and conservation strategies for local wildlife.

molecular biology↗

bioRxiv2024

A fungal transcription factor BOT6 facilitates the transition of a beneficial root fungus into an adapted anthracnose pathogen

The infection strategies employed by plant endophytes are attributed to their ability to overcome durable nonhost resistance and adapt to the host environment. However, the regulatory genetic background underlying how they adapt to the host and determine their lifestyles remains enigmatic. Here, we show that the CtBOT6, a cluster-residing transcription factor in the root-associated fungus Colletotrichum tofieldiae (Ct), plays a pivotal role in regulating virulence-related gene expression and in producing metabolites both not only within and but unexpectedly outside of the cluster. Genetic manipulation of CtBOT6 toward activation alone is sufficient to transition a root beneficial Ct along the mutualist-pathogen continuum even toward a leaf pathogen capable of overcoming nonhost resistance, partly dependent on the host abscisic acid and ethylene pathways. Our findings indicate that the status of CtBOT6 serves as a critical determinant for the endophytic fungus to adapt to the plant different environments and manifest diverse infection strategies.

plant biology↗

bioRxiv2024

Effects of foam rolling intervention on lactate clearance and cognitive function after high-intensity exercise with incremental load

PurposeThe effects foam rolling (FR) intervention on lactate clearance and the impaired executive function associated with fatigue after high-intensity exercise remain unclear. This study examined whether FR is an effective tool for fatigue recovery. MethodsEighteen healthy adults (10 males, 8 females; 21.8{+/-}1.7 years) without consistent exercise habits participated. The participants performed high-intensity exercises using an ergometer with a progressive load. Post-exercise FR intervention was compared with the control condition. Measurements included lactate, vigor/fatigue by the Profile of Mood States 2nd edition, cognitive function (cognitive task performance), and leg and body rating of perceived exertion (RPE) pre-and post-exercise, and post-intervention (5 min post-exercise). ResultsThe blood lactate concentration post-FR intervention (-7.3{+/-}3.0 mmol/L) was significantly reduced. Increased lactate clearance by FR correlated with faster recovery of executive function for those with greater lactate clearance, although cognitive fatigue was not observed after high-intensity exercise (P = .086, r = 0.41). Lactate clearance was not significantly correlated with RPE in the FR condition, whereas RPE decreased with increased lactate clearance for those with greater lactate clearance in the control condition (leg; r = 0.778, body; r = 0.669). ConclusionsThese results indicated FR intervention may be an effective recovery tool for exhausting exercises.

physiology↗

bioRxiv2024

Selective Translation Orchestrates Key Signaling Pathways in Primed Pluripotency

Pluripotent stem cell (PSC) identities, such as differentiation and infinite proliferation, have long been understood within the frameworks of transcription factor networks, epigenomes, and signal transduction, yet remain unclear and fragmented. Directing attention toward translational regulation, as a bridge between these events, promises to yield new insights into previously unexplained mechanisms. Our functional CRISPR interference screening-based approach revealed that EIF3D maintains primed pluripotency through selective translational regulation. The loss of EIF3D disrupts the balance of pluripotency-associated signaling pathways, impairing primed pluripotency. Moreover, we discovered that EIF3D ensures robust proliferation by controlling the translation of various p53 regulators, which maintain low p53 activity in the undifferentiated state. In this way, selective translation by EIF3D tunes the homeostasis of the primed pluripotency networks, ensuring the maintenance of an undifferentiated state with high proliferative potential. Therefore, this study establishes a paradigm for selective translational regulation as a defining feature of primed PSC identity.

cell biology↗

bioRxiv2023

Loss-of-function mutation in the polyamine transporter gene OsLAT5 as a selectable marker for genome editing

Genome editing by TALENs, CRISPR/Cas, base or prime editing have become routine tools. During stable plant transformation, the gene coding for the editing enzyme, e.g., Cas9, the guide RNAs (gRNAs), alongside a selectable marker are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to antibiotics or herbicides. Selectable markers use a substantial portion of the T-DNA, hence reducing transfer efficiency by limiting the effective number of TALENs or guide/pegRNAs that can be used in parallel. Moreover, marker genes are frequently subject to gene silencing. Here, we generated loss-of-function mutations in PUT/LAT-type polyamine transporter family genes to confer resistance to the phytotoxin methylviologen (MV) as a method for selection. As a proof of concept, CRISPR/Cas9 vectors with gRNAs were constructed to target three close homologs OsLAT1, OsLAT5, and OsLAT7. We show that loss of OsLAT5 (also known as OsPUT3 or OsPAR1) function was sufficient to confer resistance to MV in rice seeds, seedlings and calli, validating the editing approach of OsLAT5 to obtain a selectable marker. We discuss the potential of incorporating a gRNA cassette (for OsLAT5) as a selectable marker and a reporter for successful genome editing for optimizing editing protocols.

plant biology↗

bioRxiv2023

Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

Promiscuous editing by CRISPR/Cas systems within the human genome is a major challenge that must be addressed prior to applying these systems therapeutically. In bacteria, CRISPR/Cas systems have evolved in a co-evolutionary arms race with infectious phage viruses that contain inhibitory anti-CRISPR proteins within their genomes. Here, we harness the outcome of this co-evolutionary arms race to engineer an AcrIIA4 anti-CRISPR protein to increase the precision of CRISPR/Cas-based genome targeting. We developed an approach that specifically leveraged (1) protein language models, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements within human cells. In a single experiment, [~]10,000 AcrIIA4 variants were tested to identify lead AcrIIA4 variants that eliminated detectable off-target editing events while retaining on-target activity. The candidates were further tested in a focused round of screening that included a high-fidelity version of Cas9 as a benchmark. Finally, arrayed experiments using Cas9 delivered as ribonucleoprotein were conducted that demonstrated an increase in gene editing precision across two independent genomic loci and a reduction in the frequency of translocation events between an on-target and off-target site. Thus, language-model-guided high-throughput screening is an effective way to efficiently engineer AcrIIA4 to increase gene editing precision, which could be used to improve the fidelity of gene editing-based therapeutics and to reduce genotoxicity.

synthetic biology↗

bioRxiv2023

Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds

To survive daily damage, the formation of actomyosin ring at the wound periphery is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound are regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, we find that reduced extracellular calcium and depletion of intracellular calcium affect cell wound repair differently, despite these two conditions exhibiting similar GCaMP signals. Thus, our results suggest that, in addition to initiating repair events, both the quantity and sources of calcium influx are important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair. SummaryCells have rapid and robust repair systems to survive daily damage. This study shows that calcium influx regulates the three distinct Drosophila Annexin recruitment patterns to the cell wound in order to organize an actomyosin ring for efficient wound closure.

cell biology↗

bioRxiv2023

Two Septin Complexes Mediate Actin Dynamics During Cell Wound Repair

Cells have robust wound repair systems to prevent further damage or infection and to quickly restore cell cortex integrity when exposed to mechanical and chemical stress. Actomyosin ring formation and contraction at the wound edge are major events during closure of the plasma membrane and underlying cytoskeleton during cell wound repair. Here, we show that all five Drosophila Septins are required for efficient cell wound repair. Based on their different recruitment patterns and knockdown/mutant phenotypes, two distinct Septin complexes, Sep1-Sep2-Pnut and Sep4-Sep5-Pnut, are assembled to regulate actin ring assembly, contraction, and remodeling during the repair process. Intriguingly, we find that these two Septin complexes have different F-actin bending activities. In addition, we find that Anillin regulates the recruitment of only one of two Septin complexes upon wounding. Our results demonstrate that two functionally distinct Septin complexes work side-by-side to discretely regulate actomyosin ring dynamics during cell wound repair.

cell biology↗

bioRxiv2023

Insectivorous birds and bats outperform ants in the top-down regulation of arthropods across strata of a Japanese temperate forest

O_LIBirds, bats, and ants are recognized as significant arthropod predators. However, empirical studies reveal inconsistent trends in their relative roles in top-down control across strata. Here, we describe the differences between forest strata in the separate effects of birds, bats, and ants on arthropod communities and their cascading effects on plant damage. C_LIO_LIWe implemented a factorial design to exclude vertebrates and ants in both the canopy and understory. Additionally, we separately excluded birds and bats from the understory using diurnal and nocturnal exclosures. At the end of the experiments, we collected all arthropods and assessed herbivory damage. C_LIO_LIArthropods responded similarly to predator exclusion across forest strata, with a density increase of 81% on trees without vertebrates and 53% without both vertebrates and ants. Additionally, bird exclusion alone led to an 89% increase in arthropod density, while bat exclusion resulted in a 63% increase. Herbivory increased by 42% when vertebrates were excluded and by 35% when both vertebrates and ants were excluded. Bird exclusion alone increased herbivory damage by 28%, while the exclusion of bats showed a detectable but non-significant increase (by 22%). In contrast, ant exclusion had no significant effect on arthropod density or herbivory damage across strata. C_LIO_LIOur results reveal that the effects of birds and bats on arthropod density and herbivory damage are similar between the forest canopy and understory in this temperate forest. In addition, ants were not found to be significant predators in our system. Furthermore, birds, bats, and ants appeared to exhibit antagonistic relationships in influencing arthropod density. These findings highlight, unprecedentedly, the equal importance of birds and bats in maintaining ecological balance across different strata of a temperate forest. C_LI

ecology↗

bioRxiv2023

Deciphering Cell-types and Gene Signatures Associated with Disease Activity in Rheumatoid Arthritis using Single Cell RNA-sequencing

ObjectiveSingle cell profiling of synovial tissue has previously identified gene signatures associated with rheumatoid arthritis (RA) pathophysiology, but synovial tissue is difficult to obtain. This study leverages single cell sequencing of peripheral blood mononuclear cells (PBMCs) from patients with RA and matched healthy controls to identify disease relevant cell subsets and cell type specific signatures of disease. MethodsSingle-cell RNA sequencing (scRNAseq) was performed on peripheral blood mononuclear cells (PBMCs) from 18 RA patients and 18 matched controls, accounting for age, gender, race, and ethnicity). Samples were processed using standard CellRanger and Scanpy pipelines, pseudobulk differential gene expression analysis was performed using DESeq2, and cell-cell communication analysis using CellChat. ResultsWe identified 18 distinct PBMC subsets, including a novel IFITM3+ monocyte subset. CD4+ T effector memory cells were increased in patients with moderate to high disease activity (DAS28-CRP [≥] 3.2), while non-classical monocytes were decreased in patients with low disease activity or remission (DAS28-CRP < 3.2). Differential gene expression analysis identified RA-associated genes in IFITM3+ and non-classical monocyte subsets, and downregulation of pro-inflammatory genes in the V{delta} subset. Additionally, we identified gene signatures associated with disease activity, characterized by upregulation of pro-inflammatory genes TNF, JUN, EGR1, IFIT2, MAFB, G0S2, and downregulation of HLA-DQB1, HLA-DRB5, TNFSF13B. Notably, cell-cell communication analysis revealed upregulation of immune-associated signaling pathways, including VISTA, in patients with RA. ConclusionsWe provide a novel single-cell transcriptomics dataset of PBMCs from patients with RA, and identify insights into the systemic cellular and molecular mechanisms underlying RA disease activity.

immunology↗

bioRxiv2023

Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

plant biology↗

bioRxiv2023

Seed size control via phloem end by callose deposition/degradation of β-1,3-glucanase

Seed formation is crucial for lives of plants as well as humans; however, the mechanisms governing seed size require further investigation. Here, we present a new mechanism to modify the seed size by the newly identified phloem end that support nutrient transport, at the chalazal end of the ovule, however, blocked by callose deposition. Callose is removed after central cell fertilization (open state), allowing nutrients to be transported to the seed. However, if fertilization fails, callose deposition persists (closed state), preventing the phloem end from transporting nutrients. {beta}-1,3-glucanase genes, including putative plasmodesmata-associated proteins (AtBG_ppap), were identified as regulators of callose removal. The Atbg_ppap mutant had the phloem end in the closed state and produced smaller seeds due to incomplete callose degradation. In contrast, the AtBG_ppap overexpression line produced larger seeds than the wild type due to continuous callose degradation, indicating that the phloem end regulates substance flow via callose deposition/degradation.

plant biology↗

bioRxiv2023

Meta-analysis of gonadal transcriptomes in protogynous fishes provides novel perspective into sex change machinery

Protogyny, being capable of changing from female to male during their lifetime, is prevalent in 20 families of teleosts but is believed to have evolved within specific evolutionary lineages. Therefore, shared regulatory factors governing the sex change process are expected to be conserved across protogynous fishes. However, a comprehensive understanding of this mechanism remains elusive. To identify these factors, we conducted a meta-analysis using gonadal transcriptome data from seven species. We curated data pairs of ovarian tissue and transitional gonad, and employed ratios of expression level as a unified criterion for differential expression, enabling a meta-analysis across species. Our approach revealed that classical sex change-related genes exhibited differential expression levels between the ovary and transitional gonads, consistent with previous reports. These results validate our methodologys robustness. Additionally, we identified novel genes not previously linked to gonadal sex change in fish. Notably, changes in the expression levels of acetoacetyl-CoA synthetase and apolipoprotein Eb, which are involved in cholesterol synthesis and transport, respectively, suggest that the levels of cholesterol, a precursor of steroid hormones crucial for sex change, are decreased upon sex change onset in the gonads. This implies a potential universal influence of cholesterol dynamics on gonadal transformation in protogyny.

zoology↗

bioRxiv2023

Periodontitis promotes bacterial extracellular vesicle-induced neuroinflammation in the brain and trigeminal ganglion

Gram-negative bacteria derived extracellular vesicles (EVs), also known as outer membrane vesicles, have attracted significant attention due to their pathogenic roles in various inflammatory diseases. We recently demonstrated that EVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) can cross the blood-brain barrier (BBB) and that their extracellular RNA cargo can promote the secretion of proinflammatory cytokines, such as IL-6 and TNF-, in the brain. To gain more insight into the relationship between periodontal disease (PD) and neuroinflammatory diseases, we investigated the effect of Aa EVs in a mouse model of ligature-induced PD. When EVs were administered through intragingival injection or EV-soaked gel, proinflammatory cytokines were strongly induced in the brains of PD mice. The use of TLR (Toll-like receptor)-reporter cell lines and MyD88 knockout mice confirmed that the increased release of cytokines was triggered by Aa EVs via TLR4 and TLR8 signaling pathways and their downstream MyD88 pathway. Furthermore, the injection of EVs through the epidermis and gingiva resulted in the direct retrograde transfer of Aa EVs from axon terminals to the cell bodies of trigeminal ganglion (TG) neurons and the subsequent activation of TG neurons. We also found that the Aa EVs changed the action potential of TG neurons. These findings suggest that EVs derived from periodontopathogens such as Aa might be involved in pathogenic pathways for neuroinflammatory diseases, neuropathic pain, and other systemic inflammatory symptoms as a comorbidity of periodontitis. Author summaryExtracellular vesicles (EVs) secreted from bacterial cells play a key role in microbe-host cell communication. Bacterial EVs (bEVs) may be closely linked to the pathogenesis underlying neuroinflammatory diseases. In the current experiments, bEVs caused the brain to release proinflammatory cytokines by activating the host TLR signaling pathway. This induction of neuroinflammation was significantly more prominent in the periodontitis disease model, implying a substantial link between periodontal and neuroinflammatory diseases through bEVs. We also show that bEVs are translocated to the neuronal cell body via retrograde axonal transport, where they directly activate neuronal proinflammatory signals (Fig 8). Our findings reveal that bEVs are a pathogenic pathway for neurological conditions potentially linked to periodontitis, such as Alzheimers disease and trigeminal neuropathic pain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/548287v1_fig8.gif" ALT="Figure 8"> View larger version (32K): org.highwire.dtl.DTLVardef@ff2928org.highwire.dtl.DTLVardef@1e72688org.highwire.dtl.DTLVardef@92e002org.highwire.dtl.DTLVardef@b0e125_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig 8.C_FLOATNO Graphic summary. Model of bacterial EV (bEVs) function in the brain and trigeminal neuron. bEVs have the ability to enter host brain cells through the bloodstream and to enter the trigeminal ganglion neuronal cell body through retrograde axonal transport, where they directly trigger proinflammatory neuronal signals. Through the TLR4/TLR8-MyD88 and NF-{kappa}B signaling pathways, bEVs and their RNA cargo can stimulate the production of TNF- and IL-6 in the host cells. The production of neuroinflammation is noticeably more pronounced in the periodontitis disease model, suggesting a strong association between periodontal and neuroinflammatory diseases via bEVs. C_FIG

pathology↗